Gene gun delivery of DNA vaccines, also known as particle mediated epidermal delivery or PMED, delivers DNA vaccines coated onto 1 micron sized gold particles directly into cells of the epidermis. It achieves pain-free delivery, is considerably more efficient than electroporation or other DNA vaccine delivery approaches (requires 100-1000 fold lower doses), and induces both systemic and mucosal responses. The gene gun stands out as the only DNA vaccine technology to date that has consistently induced T cell responses and protective levels of antibody in 100% of vaccinated subjects in human clinical trials. In addition, in preclinical studies, the research grade gene gun effectively induces mucosal responses that correlate with enhanced protection mice, nonhuman primates and swine models of influenza, HSV and HIV infections. However, when transitioned into the clinic, the immunogenicity of PMED DNA vaccines in humans using a newly-designed clinical gene gun resulted in lower immune responses than what was achieved with the research device in preclinical animal models. Our preliminary studies show that these earlier clinical devices fell short of optimal engineering. In particular, the first clinical gene guns delivered the particles into a smaller area and fewer particles penetrated the skin when compared to the research device. This is likely due to the use of a polystyrene nozzle in the clinical device that generated an electrostatic charge and restricted gold particle acceleration. In addition, for both the research and clinical devices, the density distribution of the particles fell in a bell curve with the center of the target having a much higher density of particles and lower viability than the outer area. This decreased viability of the cells in the center of the target caused reduced DNA vaccine expression due to a dead center. Another obstacle to more robust vaccine expression in the use of DNA coated particles is that DNA must enter the nucleus, while the majority of delivered particles now reach only the cytoplasm and fall short of the nucleus. Here, we propose to address these limitations by incorporating novel engineering modifications to the research and clinical gene guns ? a ?spinner? apparatus that will increase the target size and particle distribution and a hybrid aluminum/plastic disposable clinical gene gun barrel to reduce electrostatic restriction of the gold particles. We will also, in collaboration with GE Global Research, investigate novel nucleic acid formulations for better nuclear localization and vaccine expression by employing a Rolling Circle Amplified (RCA) DNA and stable RNA and RNA/DNA compositions as strategies to increase the number of cells expressing the gene and the amount of protein expressed per cell. We hypothesize these modifications will result in generation of a new, more effective research and clinical gene guns with enhanced immunogenicity in vivo.